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Thursday, 24 October 2013

Key differences in the regulation of jumping genes may have arisen relatively recently in evolution

Thursday, 24 October 2013

Researchers at the Salk Institute for Biological Studies have, for the first time, taken chimpanzee and bonobo skin cells and turned them into induced pluripotent stem cells (iPSCs), a type of cell that has the ability to form any other cell or tissue in the body.

This
microscope image shows induced pluripotent

stem
cells (iPSC) from our closest living relatives.

Skin
cells from bonobos (pigmy chimps) were

reprogrammed
to pluripotent stem cells, an

advance
that allows scientists to study the

differences
between the neurons of humans and

chimps.
The colours show different aspects of the

cells'
molecular components. Credit: Courtesy of

Carol
Marchetto, Salk Institute for Biological

Studies.

Mouse iPSCs were created in 2006 by Kazutoshi Takahashi and Shinya Yamanaka at Kyoto University in Japan, and human iPSCs soon followed – feats which earned Yamanaka the Nobel Prize in Physiology or Medicine last year. Now scientists regularly use iPSCs to model diseases using cells that would be otherwise difficult to obtain from a living person or animal. By adding a combination of four key factors, a skin cell can be made into an iPSC, which can then be coaxed into forming liver, lung and brain cells in a culture dish.

It's now possible to not only model disease using the cells, but also to compare iPSCs from humans to those of our closest living relatives – great apes, with which we share a majority of genes – for insight into what molecular and cellular features make us human.

"Comparing human, chimpanzee and bonobo cells can give us clues to understand biological processes, such as infection, diseases, brain evolution, adaptation or genetic diversity," says senior research associate Iñigo Narvaiza, who led the study with senior staff scientist Carol Marchetto at the Salk Institute in La Jolla.

"Until now, the sources for chimpanzee and bonobo cells were limited to post-mortem tissue or blood. Now you could generate neurons, for example, from the three different species and compare them to test hypotheses."

In the new study, published online October 23 in the journal Nature, scientists found disparities in the regulation of jumping genes or transposons – DNA elements that can copy and paste themselves into spots throughout the genome – between humans and non-human primate cells. Jumping genes provide a means to rapidly shuffle DNA and might be shaping the evolution of our genomes, the scientists say.

From
left are Salk scientists Ahmet Denli, Carol

Marchetto,
Iñigo Narvaiza and Fred Gage.

Credit: Courtesy of the Salk
Institute forBiological Studies.

Working in the lab of Salk's Fred Gage, the Vi and John Adler Chair for Research on Age-Related Neurodegenerative Disease, Narvaiza, Marchetto and their colleagues identified genes that are differentially expressed between iPSCs from humans and both chimpanzees and bonobos.

To the group's surprise, two of those genes code for proteins that restrict a jumping gene called long interspersed element-1or L1, for short. Compared with non-human primate cells, human iPSCs expressed higher levels of these restrictors, called APOBEC3B and PIWIL2.

"We weren't expecting that," Marchetto says.

"Those genes caught our eyes, so they were the first targets we focused on."

L1 and a handful of other jumping genes are abundant throughout our genomes. Where these bits of DNA insert themselves is hard to predict, and they can produce variable effects. For example, they might completely disrupt genes, modulate them, or cause them to be processed into entirely new proteins.

Using L1 tagged with a fluorescent marker, the group observed higher numbers of fluorescent iPSCs from non-human primates compared with humans. In separate experiments, they produced iPSCs with too much or too little APOBEC3B and PIWIL2, finding – as expected – that an excess of the two proteins dampened the mobility and reduced the appearance of newly inserted DNA in the non-human primate cells.

These results suggested that L1 elements insert themselves less often throughout our genomes. Indeed, looking at genomes of humans and chimpanzees that had already been sequenced, the researchers found that the primates had more copies of L1 sequences than did humans.

The question that remains is what would be the impact of differences in L1 regulation?

"It could mean that we have gone, as humans, through one or more bottlenecks in evolution, that decrease the variability present in our genome," says Marchetto, though the hypothesis is admittedly hard to prove. It is known, however, that humans' genomes are less variable than chimpanzees'.

The new study provides proof of concept that the iPSC technology can be used to understand some of the evolutionary differences between humans and non-human primates, says Narvaiza. The group plans to make technology, and all the data, available to the broader research community – which is especially helpful now that great ape research is severely restricted in the United States and abroad – so that other scientists can learn about primates using non-invasive, ethically sound methods.

The team plans to differentiate the stem cells into other tissues, such as neurons, and comparing how the cells from different species behave. They will also use the iPSC technology to investigate how chimpanzees might differ from people in susceptibility to cancer, genetic diseases and viral infection.

Researchers at Columbia University Medical Center (CUMC) have devised a hair restoration method that can generate new human hair growth, rather than simply redistribute hair from one part of the scalp to another. The approach could significantly expand the use of hair transplantation to women with hair loss, who tend to have insufficient donor hair, as well as to men in early stages of baldness. The study was published today in the online edition of the Proceedings of the National Academy of Sciences (PNAS).

"About 90 percent of women with hair loss are not strong candidates for hair transplantation surgery because of insufficient donor hair," said co-study leader Angela M. Christiano, PhD, the Richard and Mildred Rhodebeck Professor of Dermatology and professor of genetics & development.

"This method offers the possibility of inducing large numbers of hair follicles or rejuvenating existing hair follicles, starting with cells grown from just a few hundred donor hairs. It could make hair transplantation available to individuals with a limited number of follicles, including those with female-pattern hair loss, scarring alopecia, and hair loss due to burns."

According to Dr. Christiano, such patients gain little benefit from existing hair-loss medications, which tend to slow the rate of hair loss but usually do not stimulate robust new hair growth.

"Dermal papilla cells give rise to hair follicles, and the notion of cloning hair follicles using inductive dermal papilla cells has been around for 40 years or so," said co-study leader Colin Jahoda, PhD, professor of stem cell sciences at Durham University, England, and co-director of North East England Stem Cell Institute, who is one of the early founders of the field.

Spheres of cultured papillae cells from
human

hair follicles successfully produced
new human

hair when transplanted between the
dermis and

epidermis of human skin. Credit:
Claire Higgins/

Christiano Lab at Columbia University
Medical

Center.

"However, once the dermal papilla cells are put into conventional, two-dimensional tissue culture, they revert to basic skin cells and lose their ability to produce hair follicles. So we were faced with a Catch-22: how to expand a sufficiently large number of cells for hair regeneration while retaining their inductive properties."

The researchers found a clue to overcoming this barrier in their observations of rodent hair. Rodent papillae can be easily harvested, expanded, and successfully transplanted back into rodent skin, a method pioneered by Dr. Jahoda several years ago. The main reason that rodent hair is readily transplantable, the researchers suspected, is that their dermal papillae (unlike human papillae) tend to spontaneously aggregate, or form clumps, in tissue culture. The team reasoned that these aggregations must create their own extracellular environment, which allows the papillae to interact and release signals that ultimately reprogram the recipient skin to grow new follicles.

"This suggested that if we cultured human papillae in such a way as to encourage them to aggregate the way rodent cells do spontaneously, it could create the conditions needed to induce hair growth in human skin," said first author Claire A. Higgins, PhD, associate research scientist.

To test their hypothesis, the researchers harvested dermal papillae from seven human donors and cloned the cells in tissue culture; no additional growth factors were added to the cultures. After a few days, the cultured papillae were transplanted between the dermis and epidermis of human skin that had been grafted onto the backs of mice. In five of the seven tests, the transplants resulted in new hair growth that lasted at least six weeks. DNA analysis confirmed that the new hair follicles were human and genetically matched the donors.

"This approach has the potential to transform the medical treatment of hair loss," said Dr. Christiano.

"Current hair-loss medications tend to slow the loss of hair follicles or potentially stimulate the growth of existing hairs, but they do not create new hair follicles. Neither do conventional hair transplants, which relocate a set number of hairs from the back of the scalp to the front. Our method, in contrast, has the potential to actually grow new follicles using a patient's own cells. This could greatly expand the utility of hair restoration surgery to women and to younger patients — now it is largely restricted to the treatment of male-pattern baldness in patients with stable disease."

More work needs to be done before the method can be tested in humans, according to the researchers.

"We need to establish the origins of the critical intrinsic properties of the newly induced hairs, such as their hair cycle kinetics, colour, angle, positioning, and texture" said Dr. Jahoda.

"We also need to establish the role of the host epidermal cells that the dermal papilla cells interact with, to make the new structures."

The team is optimistic that clinical trials could begin in the near future.

"We also think that this study is an important step toward the goal of creating a replacement skin that contains hair follicles for use with, for example, burn patients," said Dr Jahoda.

The researchers used gene expression analyses to determine that the three-dimensional cultures restored 22 percent of the gene expression seen in normal hair follicles. "That's less than we expected, but it was sufficient for inducing the growth of new hair follicles," said Dr. Christiano.

In addition, using methods for the analysis of regulatory networks developed by the Califano lab in the Center for Computational Biology and Bioinformatics, Department of Systems Biology, the researchers identified a number of transcription factors (gene regulators) that have the potential to mimic the environmental signals that trigger papillae to induce new hair growth. This information could help researchers develop ways to restore the expression of more genes involved in hair growth and to increase the efficiency of the induction.

Thursday, 17 October 2013

Between 2000 and 2010 in the United States the number of donor eggs used for in vitro fertilization increased, and outcomes for births from those donor eggs improved, according to a study published by JAMA. The study is being released early online to coincide with its presentation at the American Society for Reproductive Medicine and the International Federation of Fertility Societies joint annual meeting.

During the past several decades, the number of live births to women in their early 40s in the United States has increased steadily. The prevalence of oocyte (egg) donation for in vitro fertilization (IVF) has increased in the United States, but little information is available regarding maternal or infant outcomes to improve counselling and clinical decision making, according to background information in the article.

Jennifer F. Kawwass, M.D., of the Emory University School of Medicine, Atlanta, and colleagues examined trends in use of donor oocytes in the United States and assessed perinatal outcomes. The study used data from the Centers for Disease Control and Prevention's National Assisted Reproductive Technology (ART) Surveillance System (NASS); fertility centres are mandated to report their data to the system, which includes data on more than 95 percent of all IVF cycles performed in the United States. Good perinatal outcome was defined as a single live-born infant delivered at 37 weeks or later weighing 5.5 lbs. or more.

The researchers found that at 443 clinics (93 percent of all U.S. fertility centres) the annual number of donor oocyte cycles performed in the United States increased from 10,801 in 2000 to 18,306 in 2010, as did the percentage of such cycles that involved frozen oocytes or embryos (vs. fresh) (26.7 percent to 40.3 percent) and that involved elective single-embryo transfer (vs. transfer of multiple embryos) (0.8 percent to 14.5 percent). Good perinatal outcomes increased from 18.5 percent to 24.4 percent. Average age remained stable at 28 years for donors and 41 years for recipients. Recipient age was not associated with likelihood of good perinatal outcome.

"Use of donor oocytes is an increasingly common treatment for infertile women with diminished ovarian reserve for whom the likelihood of good perinatal outcome appears to be independent of recipient age. To maximize the likelihood of a good perinatal outcome, the American Society of Reproductive Medicine recommendations suggesting transfer of a single embryo in women younger than 35 years should be considered.”

“Additional studies evaluating the mechanisms by which race/ethnicity, infertility diagnosis, and day of embryo culture affect perinatal outcomes in both autologous [donor and recipient are the same person] and donor IVF pregnancies are warranted to develop preventive measures to increase the likelihood of obtaining a good perinatal outcome among ART users," the authors write.

Tuesday, 15 October 2013

An international team of researchers from the University of Copenhagen have successfully developed an innovative 3D method to grow miniature pancreas from progenitor cells. The future goal is to use this model to help in the fight against diabetes. The research results have just been published in the scientific journal Development.

The new
method allows the cell material from

mice to
grow vividly in picturesque tree-like

structures.
Credit: The Danish Stem Cell Center.

Professor Anne Grapin-Botton and her team at the Danish Stem Cell Centre have developed a three-dimensional culture method which enables the efficient expansion of pancreatic cells. The new method allows the cell material from mice to grow vividly in picturesque tree-like structures. The method offers huge long term potential in producing miniature human pancreas from human stem cells. These human miniature organs would be valuable as models to test new drugs fast and effective – and without the use of animal models.

"The new method allows the cell material to take a three-dimensional shape enabling them to multiply more freely. It’s like a plant where you use effective fertilizer, think of the laboratory like a garden and the scientist being the gardener,” says Anne Grapin-Botton.

Social cells

The cells do not thrive and develop if they are alone, and a minimum of four pancreatic cells close together is required for subsequent organoid development.

“We found that the cells of the pancreas develop better in a gel in three-dimensions than when they are attached and flattened at the bottom of a culture plate. Under optimal conditions, the initial clusters of a few cells have proliferated into 40,000 cells within a week. After growing a lot, they transform into cells that make either digestive enzymes or hormones like insulin and they self-organize into branched pancreatic organoids that are amazingly similar to the pancreas,” adds Anne Grapin-Botton.

The scientists used this system to discover that the cells of the pancreas are sensitive to their physical environment such as the stiffness of the gel and to contact with other cells.

Pancreas and diabetes connection

An effective cellular therapy for diabetes is dependent on the production of sufficient quantities of functional beta-cells. Recent studies have enabled the production of pancreatic precursors but efforts to expand these cells and differentiate them into insulin-producing beta-cells have proved a challenge.

“We think this is an important step towards the production of cells for diabetes therapy, both to produce mini-organs for drug testing and insulin-producing cells as spare parts. We show that the pancreatic cells care not only about how you feed them but need to be grown in the right physical environment. We are now trying to adapt this method to human stem cells,” adds Anne Grapin-Botton.

Saturday, 5 October 2013

A group of Brigham and Women's Hospital, and Harvard Stem Cell Institute researchers and collaborators at MIT and MGH have found a way to use stem cells as drug delivery vehicles.

Engineered mesenchymal stem cells
are

targeted to a site of inflammation to
secrete

anti-inflammatory interleukin-10 proteins.

Credit: Courtesy by
Jeffrey Karp.

The researchers inserted modified strands of messenger RNA into connective tissue stem cells — called mesenchymal stem cells — which stimulated the cells to produce adhesive surface proteins and secrete interleukin-10, an anti-inflammatory molecule. When injected into the bloodstream of a mouse, these modified human stem cells were able to target and stick to sites of inflammation and release biological agents that successfully reduced the swelling.

"If you think of a cell as a drug factory, what we're doing is targeting cell-based, drug factories to damaged or diseased tissues, where the cells can produce drugs at high enough levels to have a therapeutic effect," said research leader Jeffrey Karp, PhD, a Harvard Stem Cell Institute principal faculty member and Associate Professor at the Brigham and Women's Hospital, Harvard Medical School and Affiliate faculty at MIT.

Karp's proof of concept study, published in the journal Blood, is drawing early interest from biopharmaceutical companies for its potential to target biological drugs to disease sites. While ranked as the top sellers in the drug industry, biological drugs are still challenging to use, and Karp's approach may improve their clinical application as well as improve the historically mixed, clinical trial results of mesenchymal stem cell-based treatments.

This is Jeffrey Karp, Ph.D., a Harvard
Stem

Cell Institute principal faculty member
and

associate professor at the Brigham and

Women's Hospital, Harvard Medical
School

and Affiliate faculty at MIT. Credit:

Brigham and Women's Hospital.

Mesenchymal stem cells have become cell therapy researchers' tool of choice because they can evade the immune system, and thus are safe to use even if they are derived from another person. The researchers' method of engineering the cells with messenger RNA is also harmless, as it cannot integrate into a cell's genome, which can be a problem when DNA is used (via viruses) to manipulate gene expression.

"This opens the door to thinking of messenger RNA transfection of cell populations as next generation therapeutics in the clinic, as they get around some of the delivery challenges that have been encountered with biological agents", said Oren Levy, co-lead author of the study and Instructor of Medicine in Karp's lab. The study was also co-led by Professor Weian Zhao at University of California, Irvine who was previously a postdoctoral fellow in Karp's lab.

One such challenge with using mesenchymal stem cells is they have a "hit-and-run" effect, since they are rapidly cleared after entering the bloodstream, typically within a few hours or days. The Harvard/MIT team demonstrated that rapid targeting of the cells to the inflamed tissue produced a therapeutic effect despite the cells being rapidly cleared. The scientists want to extend cell lifespan even further and are experimenting with how to use messenger RNA to make the stem cells produce pro-survival factors.

"We're interested to explore the platform nature of this approach and see what potential limitations it may have or how far we can actually push it," Zhao said. "Potentially, we can simultaneously deliver proteins that have synergistic therapeutic impacts."